Short pulses in optical microscopy Ivan Scheblykin, Chemical Physics, LU

1. Short pulses in optical microscopy Ivan Scheblykin, Chemical Physics, LU Outline: Introduction to traditional optical microscopy based on single photon absorption: Fluorescence wide-field and conforcal microscopy Introduction to single molecule imaging 2-photon absorption 2-photon confocal fluorescence microscopy 3-photon absorption, second harmonic generation Microscopy which is not limited by light diffraction

2. Why do we see objects ? Changing of the properties of light coming to the sample: Light absorption Light scattering Changing of light polarization … An object emits light itself: Luminescence Second-harmonic generation ….. Many different ways to create contrast in optical microscopy

3. object Transmission image Absorption and scattering

4. 100€ object object object Transmission image Absorption and scattering Excitation light Blocking filter

5. 100€ object object Transmission image Absorption and scattering Sample is stained by a fluorescent dye Excitation light Fluorescence Blocking filter

7. White board Microscope scheme

9. Numerical Apreture Spherical angle S Light collection efficiency S/4 NA/n = 1 , 50% NA/n = 0.6, 10% 

12. Confocal fluorescence microscope Wide-field fluorescence microscope 10 microns

14. 3D imaging, z-scan

15. Single molecule spectroscopy Can we see one single chromophore ? Not in absorption, because cross section is too small  = 10-16 cm2 , 10-8 cm = 0.1 nm However, we can detect fluorescence light emitted by the molecule!

16. 5  Sample For SMS

17. Single molecule imaging Chemical Physics, Single Molecule Spectroscopy group, LU

18. Other ways to create contrast Non-linear processes induced by strong laser light Observation of fluorescence excitated by 2-photon absorption 3-photon absorption Observation of second harmonic signal third harmonic signal Absorption, scattering

19. Two-photon absorption Theory - Maria Göppert-Mayer, 1929 Experimental observation – 1961 Using in microscopy – Denk, Strickler, Webb, Science 1990 Probability of excitaion (W)  (Intensity)2 W  ( I[ptonots/cm2/s] )2 f Virtual level Absorbed photon Fluorescence i

20. One and Two-photon absorption cross sections Transition dipole moment moment Estimation of 2 (WB)

22. Two-photon excitation versus one-photon excitation Dye solution, safranin O 543 nm excitation 1046 nm excitation

23. Resolution of 2-photon microscopy XY, Z, 1/z4 excitation probability dependence And 1/z2 dependence of total fluorescence (WB)

25. The same fluorescence signal from the sample I2 I1

27. Some advantages of 2-photon excitation versus one-excitation in confocal microscopy Better Light collection efficiency.. Multi-photon excitation confines fluorescence excitation to a small volume at the focus of the objective. Photon flux is insufficient in out-of-focus planes to excite fluorescence. No confocal pinhole is needed. All fluorescence (even scattered photons) constitutes useful signal. Photobleaching and photodamage are limited to the zone of 2P excitation and do not occur above or beyond the focus. Larger penetration depth. IR photons travel deeper into tissue with less scattering and absorption comparing to visible photons. Scattering 1/4! In practice - approximaterly 2 times larger penetration depth. Much smaller background from impurity fluorescence when IR laser is used in comparison with VIS or UV light. 2 photon excitation spectra are usually very broad. Therefore, one laser source can be used for many different dyes having different fluorescence wavelengths. No chromatic aberration problems.

28. Even scattered fluorescence photons are usefull in 2-photon regime

30. All the dyes are excited by the same laser! No effect of chtomatic aberration (White board)

32. Other ways to create contrast Non-linear processes induced by strong laser light Observation of fluorescence excitated by 2-photon absorption 3-photon absorption Absorption, scattering Observation of second harmonic signal third harmonic signal SHG microscopy is generally used to observe non-centrosymmetric structures SHG is forbidden where there is an inversion symmetry, and this constraint makes it a sensitive tool for the study of interfaces and surfaces One can get a signal even without using any dyes to stain the sample SHG is cohherent processes: Intensity  N2 Fluorescence is noncohherent processes: Intensity  N Number of molecules Cross-section of SHG on a molecules is very small, but collective response from many molecules can compensate it !

33. Third harmonic generation image, No dye staining was applied

34. Optical microscopy beyond diffraction limit ????? Diffraction limit – distribution of light intensity However, if the process is nonlinear function of intensity, then the localization is not limited by the wavelength

35. Excited state depletion Excitation pulse S1 Excitation pulse Fluorescence S0

36. Excited state depletion STED pulse Excitation pulse Stimulated emission S1 Excitation pulse Stimulated emission Fluorescence S0

37. Excited state depletion STED pulse Excitation pulse Stimulated emission S1 Photons in STED pulse has lower energy to avoid excitation. Pulse duration should much shorter then S1 lifetime = 1/Kfluores Excitation pulse Stimulated emission Kinternal relaxation >KSM>> Kfluorescence Fluorescence is completely suppressed by stimulated emission process. S0 Suturation condition for STED pulse: KSM=Kfluorescence ; Isaturationabsorption~ 1 ns-1

38. Imax>> Isaturation Fluorescence f(x) - Spatial distribution of the STED pulse Saturation parameter:  = I max/ Isaturation f(x) = sin2(2/) x= /100, when =1000 Excitation spot x ~